Thyroid hormones, as well as retinoic acid (RA) function through multiple nuclear receptors that belong to the steroid/thyroid hormone receptor superfamily (reviewed by Evans 1988; Green and Chambon, 1988). The thyroid hormone receptors (TR) are encoded by two genes (Weinberger et al., 1986; Jansson et al., 1983), referred to as TRα and TRβ from which multiple isoforms are generated (Benbrook and Pfahl, 1987; Nakai et al., 1988; Mitsuhashi et al., 1988; Lazar et al., 1989; Koenig et al., 1989; Sakurai et al., 1989; Hodin et al., 1989). The known TRα subtypes are generated by alternative mRNA splicing yielding several isoforms with distinct carboxyterminal regions (Sap et al., 1986; Benbrook and Pfahl, 1987; Thompson et al., 1987; Mitsuhashi et al, 1988; Nakai et al., 1988). Only one of these isoforms, TRα-1, is a classical ligand dependent transcriptional activator, while for the other splicing variants (TRα-2 and TRα-2V) a function as transcriptional activator could not be demonstrated (Mitsuhashi et al, 1988; Lazar et al., 1989; Koenig et al., 1989; Schueler et al., 1990; Hermann et al., 1991). Although TRα-2 has been shown to exhibit weak repressor activity (Lazar et al., 1989; Koenig et al., 1989; Hermann et al., 1991), the biological functions of the carboxyterminal TRα variants are not well understood. Two TRβ forms have been described (Weinberger et al., 1986; Hodin et al., 1989) that differ in their amino terminal regions and both are transcriptional activators. Besides their classical roles as ligand dependent enhancer proteins, TRα-1 and TRβ-1 function as transcriptional repressors and/or silencer proteins in the absence of ligand (Graupner et al., 1989; Damm et al., 1989; Zhang et al., 1991b; Brent et al., 1989; Graupner et al., 1991; Baniahmad et al., 1990).
Retinoic acid receptors (RAR) are encoded by three genes RARα, β, and γ (Petkovich et al., 1987; Giguere et al., 1987; Benbrook et al., 1988; Krust et al., 1989) from which multiple isoforms that differ in their amino terminal regions, are generated by a combination of alternative promoter usage and alternative splicing (Zelent et al., 1991; Lehmann et al., 1991; Leroy et al., 1991). All RAR isoforms can antagonize each other's activity (Husmann et al., 1991). A second type of RA receptor was more recently described that is only activated by high concentrations of RA and does not show significant homology in its ligand binding domain with RAR but has significant homology in its DNA binding domain (Mangelsdorf et al., 1990). It has been proposed that this receptor may be activated by an unknown RA metabolite derivative (Mangelsdorf et al., 1990) and it has been designated retinoid x receptor (RXRα). This receptor is highly homologous to a previously isolated orphan receptor H-2 RIIBP (Hamada et al., 1989) now usually referred to as RXRβ.
TRs as well as the retinoid receptors are believed to function as dimeric or multimeric proteins since they recognize and bind specifically to dimeric or multimeric response elements, that are either direct repeats or palindromic repeats. Certain response elements like the palindromic TRE, are activated by all three types of receptors, TRs, RARs, and RXRs (Umesono et al., 1988; Graupner et al., 1989; Mangelsdorf et al., 1990) while other response elements are receptor specific (Hoffmann et al., 1990; Umesono et al., 1991; Näär et al., 1991). A direct repeat of the sequence TGACCT can function as a specific response element for TRs, RARs and vitamin D receptors depending on whether the repeats are separated by 4, 5 or 3 spacer nucleotides, respectively (Umesono et al., 1991). However, spacing between half-sites of response elements does not solely determine receptor specificity (Näär et al., 1991; our unpublished results).
Although a large set of data appears to suggest that TRs and RARs function as homodimers, there exists no convincing experimental evidence yet that these proteins interact with their responsive elements in vivo or in vitro specifically as homodimers. To the contrary, an increasing volume of data suggests that TRs as well as RARs require accessory nuclear proteins for efficient DNA binding (Lazar and Berrodin, 1990; Glass et al., 1990; Murray and Towle, 1989; Burnside et al., 1990; Zhang et al., 1991a), consistent with recent data from others (Forman and Samuels, 1991). Deletion of a portion of the TRα carboxyterminal region appears to increase DNA binding and greatly enhances dimerization and/or oligomerization, suggesting that one dimerization domain of TRα is located in the “DNA binding domain” (DBD). This concept is supported by structural data on the glucocorticoid (GR) (Härd et al., 1990; Luisi et al., 1991) and estrogen (ER) receptors (Schwabe et al., 1990). A second dimerization/oligomerization domain was found to be located in the “ligand binding domain” (LBD), a region that has been suggested to form a leucine zipper type structure (Forman et al., 1989). Part of the carboxyterminal region appears to inhibit the dimerization function of TRα such that homodimers with a palindromic TRE are not efficiently formed (Zhang et al., 1991a). Enhancement of DNA binding and the formation of a slow electrophoretic mobility complex required the presence of a protein present in nuclear extracts from a number of cell lines including F9 cells, CV-1 cells, and GC cells (Zhang et al., 1991a).
The nature of this protein could not be determined, however it is reasonable to hypothesize that this protein(s) and/or the proteins that interact with TRs and RARs, as described by others (Lazar and Berrodin, 1990; Glass et al., 1990; Murray and Towle, 1989; Burnside et al., 1990; Rosen et al., 1991) are important components for these nuclear receptors that regulate their activity. Whether the protein(s) are members of the nuclear receptor family is not yet known, however we present data in this publication that one of the retinoid receptors, RXRα, strongly enhances binding of TRs and RARs to several response elements. Studies of the enhanced and upshifted TR or RAR complexes by antibodies and receptor mutants demonstrate that RXRα can form a heterodimer with TRs and RARs. The interaction can occur in the absence of DNA and requires both DNA and ligand binding domains of RXRα and the ligand binding domain of TRs or RARs. In cotransfection experiments, RXRα greatly enhances TR and RAR transcriptional activation activity at retinoic acid concentrations where RXRα itself is not significantly activated. Our data suggest that RXRα belongs to a novel class of nuclear receptors that we would like to term “booster receptors” (B-receptors) that at low ligand concentrations greatly enhance the activity of other receptors by heterodimer formation while, when by themselves, can not dimerize efficiently and have only low affinity for their ligands.